The present invention relates to an assay for the detection of sodium channel-affecting toxins, particularly marine toxins, based upon mitochondrial dehydrogenase activity in the presence of ouabain and veratridine. More specifically, the present invention concerns a cell bioassay that allows the detection of either sodium channel-blocking toxins, such as saxitoxin, or sodium channel-activating toxins, such as brevetoxin or ciguatoxin.
Commercially important species of shellfish and finfish are known to occasionally present a serious health risk to consumers due to the presence of accumulated marine toxins. A significant number of these marine toxins exert their effects by interaction with voltage-sensitive sodium channels in excitable membranes. These toxins may be sodium channel-blocking toxins or sodium channel-activating toxins. For example, paralytic shellfish poisoning (PSP) is attributed to the ingestion of molluscan shellfish that have accumulated saxitoxins, which selectively block ion transport at the sodium channel, or related compounds from toxic dinoflagellate blooms. Neurotoxic shellfish poisoning (NSP) is caused by the ingestion of shellfish that have sequestered brevetoxins from the dinoflagellate associated with Florida's red tide. Brevetoxins perturb normal membrane properties of excitable cells by activating sodium channels. Another class of marine neurotoxins, ciguatoxins, a group of structurally related polyethers which accumulate in tropical fish, exert their biological effects through the activation of the sodium channel.
Monitoring programs for marine toxins have depended in large part upon mouse bioassays. Although mouse bioassays have for many years provided a fairly reliable assessment of risk, there is mounting pressure to develop alternative assays to reduce the reliance on animal testing. To this end, Kogure et al. developed a tissue culture assay for sodium channel-blocking toxins such as tetrodotoxin and saxitoxin. Kogure et al., Toxicon 26 (2): 191-97 (1988). In the Kogure assay, a mouse neuroblastoma cell line (Neuro-2a) is treated with a fixed concentration of the sodium channel-activator veratridine in the presence of ouabain, an inhibitor of Na+/K+ ATPase. The combined effect of these agents is an enhanced sodium influx, leading to altered cell morphology, subsequent decrease in cell viability and ultimate cell lysis. Tetrodotoxin, saxitoxin and related toxins which block sodium channels antagonize this effect, essentially "rescuing" the cells in a dose-dependent manner. This phenomenon provides the basis of a sensitive in vitro bioassay for these toxins. Evaluation of the Kogure assay requires the visual scoring of 200 or more cells per sample or well, which makes this assay a potentially time-consuming and operator-dependent task.
Scoring of this assay was improved by the modifications described by Jellett et al., Toxicon, 30 (10): 1143-56 (1992), the contents of which are hereby incorporated by reference. Jellett et al. used a microplate reader for automated determinations of absorbances of toxin-treated cells which were stained with crystal violet. This assay exploits the difference in adherence to the culture well of cells treated only with ouabain/veratridine and PSP toxin-treated cells. The former cells exhibit diminished adherence to the culture well, associated with swelling and lysis, and are readily removed by rinsing, whereas the latter cells which are protected from the effects of ouabain/veratridine, retain substrate adherence. Thus, cells affected only by ouabain/veratridine lose adherence and are removed during rinsing, while cells inoculated with the toxin remain in the well.
In the Jellett assay, wells containing Neuro-2a cells are inoculated with toxin and then with ouabain/veratridine, incubated, and subsequently rinsed. After rinsing, the wells are fixed and stained with crystal violet. The processed plates are then dried, and the stained cells are digested in acetic acid. Finally, the plates are read for absorbance of crystal violet in each well, with the absorbance being directly related to the amount of PSP toxin originally present. While these modifications notably improve the cell bioassay of Kogure et al., the assay still requires numerous steps and involves the mechanical removal of cells and treatment of the plates which are subject to operator variability.
There is a need, therefore, for a simplified bioassay for detecting sodium channel-blocking toxins. There also is a need for a tissue culture-based bioassay which can detect sodium channel-activating toxins. There also is a need for a tissue culture-based bioassay that is amenable to detecting both sodium channel-blocking and sodium channel-activating toxins, as well as an assay for determining the sodium channel affect of a toxin.